Microbial consortia

ABSTRACT

Disclosed herein are microbial consortia and compositions including microbes, for example, for use in agricultural or biodegradation applications. In some embodiments, soil, plants, and/or plant parts (such as seeds, seedlings, shoots, roots, leaves, fruit, stems, or branches) are contacted with a disclosed microbial consortia or composition including microbes. The microbial consortia or microbe-containing compositions may be applied to soil, plant, and/or plant parts alone or in combination with additional components (such as chitin, chitosan, glucosamine, amino acids, and/or liquid fertilizer). In additional embodiments, the disclosed microbial consortia or compositions including microbes are used in methods of degrading biological materials, such as chitin-containing biological materials.

CROSS REFERENCE TO RELATED APPLICATIONS

This claims the benefit of U.S. Provisional Application No. 62/126,337,filed Feb. 27, 2015, which is incorporated herein by reference in itsentirety.

FIELD

This disclosure relates to microbial consortia and methods of use of themicrobes included in the consortia, particularly for biodegradation andagricultural processes and uses.

BACKGROUND

World food demand continues to increase under pressure of increasingpopulation growth. However, agricultural workers are faced withshrinking amounts of land available for agriculture, soil depletion, andchanging environmental conditions, among other challenges. Thus, thereis a need to develop compositions and techniques that can increase foodproduction, while also decreasing the use of potentially harmfulherbicides, insecticides, and fungicides.

SUMMARY

Disclosed herein are microbial consortia and compositions includingmicrobes for use in agricultural or biodegradation applications. In someembodiments, a microbial composition of the present disclosure is themicrobial consortium deposited with the American Type Culture Collection(ATCC, Manassas, Va.) on Nov. 25, 2014, and assigned deposit numberPTA-121751 (also referred to herein as A1002) or a composition includingsome or all of the microbes in A1002. In other embodiments, acomposition of the present disclosure includes microbes from five ormore microbial species selected from Bacillus spp., Azospirillum spp.,Pseudomonas spp., Lactobacillus spp., Desulfococcus spp.,Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp.Ruminococcus spp., Aquabacterium spp., Acidisoma spp., Microcoleus spp.,Clostridium spp., Xenococcus spp., Brevibacterium spp., and Methanosaetaspp. In additional embodiments, the composition includes microbes fromfive or more (such as 5, 10, 15, or more) of the microbes listed inTable 1. The disclosed compositions may also include additionalcomponents, including but not limited to one or more of additionalmicrobe species chitin, chitosan, glucosamine, and/or amino acids.

Also disclosed are agricultural uses of the disclosed microbialconsortia or compositions. In some embodiments, the methods (uses)include contacting soil, plants, and/or plant parts (such as seeds,seedlings, roots, leaves, stems, or branches) with a disclosed microbialconsortium (such as A1002), a composition including some or all of themicrobes from A1002, or a composition including five or more of themicrobial species listed in Table 1. The microbial consortia ormicrobe-containing compositions may be applied to soil, plant, and/orplant parts alone or in combination with additional components (such asadditional microbes, chitin, chitosan, glucosamine, protein, aminoacids, and/or soil supplements or fertilizer, such as liquidfertilizer).

In additional embodiments, the disclosed microbial consortia orcompositions including microbes are used in methods of degradingbiological materials, such as chitin-containing biological materials. Insome examples, the chitin-containing materials are mixed with amicrobial consortium (such as A1002) or a composition including five ormore of the microbial species listed in Table 1 and fermented to producea fermented mixture. The fermented mixture optionally may be separatedinto solid and liquid fractions. The fermented mixture or fractionsproduced therefrom can be used in agricultural applications incombination with the disclosed microbial consortia or compositions, orcan be used in further degradation processes, for example to produceincreased levels of degradation products in the fractions.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing an exemplary fermentation process used toobtain the A1002 microbial consortium.

FIG. 2 is a schematic showing an exemplary process for biodegradation ofa chitin-containing biological material (exemplified as shrimp waste)with a disclosed microbial consortium or microbial composition.

FIG. 3 is a schematic showing an exemplary process for biodegradation ofchitin with a disclosed microbial consortium or microbial composition(such as A1002).

FIGS. 4A-4G are graphs showing the effect on yield of treatment of cornwith a microbial composition (FIGS. 4A-4C and 4E), HYTb (FIGS. 4D and4F), or a microbial composition under water stress conditions (FIG. 4G).

FIGS. 5A-5D show the effect of treatment of wheat with a microbialcomposition (FIGS. 5A-5B) or with a microbial composition plus HYTb(FIG. 5C) on yield. FIG. 5D is a digital image showing roots of wheatplants treated with a microbial composition plus HYTb (test) compared tocontrol plants.

FIGS. 6A-6E are a series of graphs showing the effect on yield oftreatment of tomato with a microbial composition.

FIG. 7 is a graph showing the effect on yield of treatment of sunflowerwith a microbial composition.

FIG. 8 is a graph showing the effect on yield of treatment of rice witha microbial composition.

FIGS. 9A-9B show the effect on yield of treatment of soybean with amicrobial composition (FIG. 9A) or with a microbial composition plusHYTb (FIG. 9B).

FIG. 10 is a graph showing the effect on yield of treatment ofstrawberry with a microbial composition plus HYTb.

FIG. 11 is a graph showing the effect on yield of treatment of beetrootwith a microbial composition plus HYTb.

FIGS. 12A and 12B are graphs showing the effect on yield of treatment ofgreen cabbage with a microbial composition plus HYTb in two trials(FIGS. 12A and 12B, respectively).

FIG. 13 is a graph of a cucumber vigor assay showing first leaf areaindex on day 18 in plants treated with HYTa (A1002). *p<0.01 by ANOVAanalysis.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases and amino acids, as defined in 37C.F.R. § 1.822. In at least some cases, only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

SEQ ID NOs: 1 and 2 are forward and reverse primers, respectively, usedto amplify 16S rDNA from A1002.

DETAILED DESCRIPTION

In nature, the balance of microbial species in the soil is influenced bysoil type, soil fertility, moisture, competing microbes, and plants(Lakshmanan et al., Plant Physiol. 166:689-700 2014). The interplaybetween microbial species and plants is further affected by agriculturalpractices, which can improve or degrade the soil microbiome (Adair etal., Environ. Microbiol. Rep. 5:404-413 2013; Carbonetto et al., PLoSOne 9:e99949 2014; Ikeda et al., Microbes Environ. 29:50-59 2014).Fertile or highly productive soils contain a different composition ofnative microbes than soil that is depleted of nutrients and linked tolow crop productivity. Different microbial species are associatedclosely with plants, on the above ground plant surfaces in thephyllosphere, at the root surface in the soil rhizosphere, or intimatelyas endophytes. Large-scale DNA analysis of these microbe associationshas revealed unexpected phylogenetic complexity (Rincon-Florez et al.,Diversity 5:581-612 2013; Lakshmanan et al., Plant Physiol. 166:689-7002014). Studies have determined complex microbiomes can be correlated toplant productivity, crop yield, stress tolerance, secondary metaboliteaccumulation, and disease tolerance (Bhardwaj et al., Microbial CellFactories 13:66-75, 2014; Vacheron et al., Frontiers Plant Science4:1-19 2014). Furthermore, plants can specifically select the microbialmixtures from the local environment and potentially fine-tune themicrobiome at the level of crop variety (Hartmann et al., Plant Soil321:235-257 2009; Doornbos et al., Agron. Sustain. Dev. 32:227-243 2012;Marasco et al., PLoS One 7:e48479 2012; Peiffer et al., Proc. Natl.Acad. Sci. USA 110:6548-6553; Bulgarelli et al., Ann. Rev. Plant Biol.64:807-838 2014).

Root-associated microbes can promote plant and root growth by promotingnutrient cycling and acquisition, by direct phytostimulation, bymediating biofertilization, or by offering growth advantage throughbiocontrol of pathogens. Agriculturally useful populations include plantgrowth promoting rhizobacteria (PGPR), pathogen-suppressive bacteria,mycorrhizae, nitrogen-fixing cyanobacteria, stress tolerance endophytes,plus microbes with a range of biodegradative capabilities. Microbesinvolved in nitrogen cycling include the nitrogen-fixing Azotobacter andBradyrhizobium genera, nitrogen-fixing cyanobacteria, ammonia-oxidizingbacteria (e.g., the genera Nitrosomonas and Nitrospira),nitrite-oxidizing genera such as Nitrospira and Nitrobacter, andheterotrophic-denitrifying bacteria (e.g., Pseudomonas and Azospirillumgenera; Isobe and Ohte, Microbes Environ. 29:4-16 2014). Bacteriareported to be active in solubilization and increasing plant access tophosphorus include the Pseudomonas, Bacillus, Micrococcus, andFlavobacterium, plus a number of fungal genera (Pindi et al., J.Biofertil. Biopest. 3:4 2012), while Bacillus and Clostridium specieshelp solubilize and mobilize potassium (Mohammadi et al., J. Agric.Biol. Sci. 7:307-316 2012). Phytostimulation of plant growth and reliefof biotic and abiotic stresses is delivered by numerous bacterial andfungal associations, directly through the production of stimulatorysecondary metabolites or indirectly by triggering low-level plantdefense responses (Gaiero et al., Amer. J. Bot. 100:1738-1750 2013;Bhardwaj et al., Microbial Cell Factories 13:66-76 2014).

In addition to activity in the environment, microbes can also deliverunique biodegradative properties in vitro, under conditions of directedfermentation. Use of specific microbial mixtures to degrade chitin andtotal protein can yield new bioactive molecules such as free L-aminoacids, L-peptides, chitin, and chitosan known to enhance growth or booststress tolerance via activation of plant innate immunity (Hill et al.,PLoS One 6:e19220 2011; Tanaka et al., Plant Signal Behav. E22598-1472013). Specific microbial communities can serve multiple tasks, bydelivering unique fermentation breakdown products, which are themselvesbiologically beneficial to crops, plus the resultant microbialconsortium, which can be delivered as an agricultural product to enhancecrop productivity.

As described herein, consortia of aerobic and/or anaerobic microbesderived from fertile soil and marine sources have been successfullyco-fermented and stabilized, offering direct growth and yield benefitsto crops. Enzymatic activity of these microbial mixtures has furtheryielded fermentation products with chitin, glucosamine, protein, and/oramino acids. In some embodiments, direct delivery of microbial consortiaand/or compositions can allow early root colonization and promoterhizosphere or endophytic associations. In some embodiments, benefits ofdelivery of microbial consortia to plants include one or more ofincreased root growth, increase root hair production, increased rootsurface area, stronger plants able to withstand transplantation shock,faster stand establishment, resistance to abiotic stress, and higherplant productivity and yield. Complex microbial mixes can span acrossplant species and genotypes, interacting with microbial soil communitiesto offer benefits to a wide range of crops growing under differentagricultural conditions.

I. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Krebs et al., Lewin's Genes XI, published by Jones andBartlett Learning, 2012 (ISBN 1449659853); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Publishers,1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 2011 (ISBN 8126531789); and George P. Rédei, EncyclopedicDictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003(ISBN: 0-471-26821-6).

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art to practice the present disclosure. The singular forms “a,”“an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a cell”includes single or plural cells and is considered equivalent to thephrase “comprising at least one cell.” As used herein, “comprises” means“includes.” Thus, “comprising A or B,” means “including A, B, or A andB,” without excluding additional elements. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety for all purposes. In case ofconflict, the present specification, including explanations of terms,will control.

Although methods and materials similar or equivalent to those describedherein can be used to practice or test the disclosed technology,suitable methods and materials are described below. The materials,methods, and examples are illustrative only and not intended to belimiting.

To facilitate review of the various embodiments of this disclosure, thefollowing explanations of specific terms are provided:

Aquatic Animal: An animal that lives in salt or fresh water. Inparticular embodiments disclosed herein, an aquatic animal includesaquatic arthropods, such as shrimp, krill, copepods, barnacles, crab,lobsters, and crayfish. In other embodiments, an aquatic animal includesfish. An aquatic animal by-product includes any part of an aquaticanimal, particularly parts resulting from commercial processing of anaquatic animal. Thus, in some examples, aquatic animal by-productsinclude one or more of shrimp cephalothorax or exoskeleton, crab orlobster exoskeleton, or fish skin or scales.

Contacting: Placement in direct physical association, including both insolid and liquid form. For example, contacting can occur with one ormore microbes (such as the microbes in a microbial consortium) and abiological sample in solution. Contacting can also occur with one ormore microbes (such as the microbes in a microbial consortium) and soil,plants, and/or plant parts (such as foliage, stem, seedling, roots,and/or seeds).

Culturing: Intentional growth of one or more organisms or cells in thepresence of assimilable sources of carbon, nitrogen and mineral salts.In an example, such growth can take place in a solid or semi-solidnutritive medium, or in a liquid medium in which the nutrients aredissolved or suspended. In a further example, the culturing may takeplace on a surface or by submerged culture. The nutritive medium can becomposed of complex nutrients or can be chemically defined.

Fermenting: A process that results in the breakdown of complex organiccompounds into simpler compounds, for example by microbial cells (suchas bacteria and/or fungi). The fermentation process may occur underaerobic conditions, anaerobic conditions, or both (for example, in alarge volume where some portions are aerobic and other portions areanaerobic). In some non-limiting embodiments, fermenting includes theenzymatic and/or non-enzymatic breakdown of compounds present in aquaticanimals or animal by-products, such as chitin.

Liquid fertilizer: An aqueous solution or suspension containing solublenitrogen. In some examples, the soluble nitrogen in a liquid fertilizerincludes an organic source of nitrogen such as urea, or urea derivedfrom anhydrous ammonia (such as a solution of urea and ammonium nitrate(UAN)). Aqua ammonia (20-32% anhydrous ammonia) can also be used. Inother examples, the soluble nitrogen in a liquid fertilizer includesnitrogen-containing inorganic salts such as ammonium hydroxide, ammoniumnitrate, ammonium sulfate, ammonium pyrophosphate, ammonium thiosulfateor combinations of two or more thereof. In some embodiments the liquidfertilizer includes a non-naturally occurring nitrogen source (such asammonium pyrophosphate or ammonium thiosulfate) and/or othernon-naturally occurring components.

Common liquid non-natural fertilizer blends are specified by theircontent of nitrogen-phosphate-potassium (N-P-K percentages) and includeaddition of other components, such as sulfur or zinc. Examples ofhuman-made blends include 10-34-0, 10-30-0 with 2% sulfur and 0.25% zinc(chelated), 11-37-0, 12-30-0 with 3% sulfur, 2-4-12, 2-6-12, 4-10-10,3-18-6, 7-22-5, 8-25-3, 15-15-3, 17-17-0 with 2% sulfur, 18-18-0,18-18-0 with 2% sulfur, 28-0-0 UAN, 9-27-0 with 2% sulfur and potassiumthio-sulfate.

Microbe: A microorganism, including but not limited to bacteria,archaeabacteria, fungi, and algae (such as microalgae). In someexamples, microbes are single-cellular organisms (for example, bacteria,cyanobacteria, some fungi, or some algae). In other examples, the termmicrobes includes multi-cellular organisms, such as certain fungi oralgae (for example, multicellular filamentous fungi or multicellularalgae).

Microbial composition: A composition (which can be solid, liquid, or atleast partially both) that includes at least one microbe (or apopulation of at least one microbe). In some examples, a microbialcomposition is one or more microbes (or one or more populations ofmicrobes) in a liquid medium (such as a storage, culture, orfermentation medium), for example, as a suspension in the liquid medium.In other examples, a microbial composition is one or more microbes (orone or more populations of microbes) on the surface of or embedded in asolid or gelatinous medium (including but not limited to a cultureplate), or a slurry or paste.

Microbial consortium: A mixture, association, or assemblage of two ormore microbial species, which in some instances are in physical contactwith one another. The microbes in a consortium may affect one another bydirect physical contact or through biochemical interactions, or both.For example, microbes in a consortium may exchange nutrients,metabolites, or gases with one another. Thus, in some examples, at leastsome of the microbes in a consortium may be metabolicallyinterdependent. Such interdependent interactions may change in characterand extent through time and with changing culture conditions.

II. Microbial Consortia and Compositions

Disclosed herein are several microbial consortia. An exemplary microbialconsortium of the present disclosure was deposited with the AmericanType Culture Collection (ATCC, Manassas, Va.) on Nov. 25, 2014, andassigned deposit number PTA-121751, referred to herein as A1002. TheA1002 consortium includes at least Bacillus spp., Azospirillum spp.,Pseudomonas spp., Lactobacillus spp., Desulfococcus spp.,Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp.Ruminococcus spp., Aquabacterium spp., Acidisoma spp., Microcoleus spp.,Clostridium spp., Xenococcus spp., Brevibacterium spp., Methanosaetaspp., Lysinibacillus spp., and Paenibacillus spp., identified bymicroarray analysis and/or 16S rDNA sequencing. Also disclosed hereinare consortia or microbial compositions including two or more (such as 2or more, 5 or more, 10 or more, 20 or more, or 50 or more) or all of themicrobes in A1002. In some embodiments, a microbial compositiondisclosed herein is a defined composition, for example a compositionincluding specified microbial species and optionally, additionalnon-microbial components (including but not limited to, salts, traceelements, chitin, chitosan, glucosamine, and/or amino acids).

As discussed below, the identity of microbes present in A1002 wasdetermined using microarray analysis (Example 3) and/or 16S rDNAsequencing (Example 4). Additional techniques for identifying microbespresent in a microbial mixture or consortium are known to one ofordinary skill in the art, including sequencing or PCR analysis ofnucleic acids, such as 16S rDNA, from individual microbial coloniesgrown from within the consortium or mixture. Additional techniques foridentifying microbes present in a microbial mixture or consortium alsoinclude 1) nucleic acid-based methods which are based on the analysisand differentiation of microbial DNA (such as DNA microarray analysis ofnucleic acids, metagenomics or in situ hybridization coupled withfluorescent-activated cell sorting (FACS)), 2) biochemical methods whichrely on separation and identification of a range biomolecules includingfatty acid methyl esters analysis (FAME), Matrix-assisted laserdesorption ionization-time of flight (MALDI-TOF) mass spectrometryanalysis, or cellular mycolic acid analysis by High Performance LiquidChromatography (MYCO-LCS) analysis, and 3) microbiological methods whichrely on traditional tools (such as selective growth and microscopicexamination) to provide more general characteristics of the community asa whole, and/or narrow down and identify only a small subset of themembers of that community.

In some examples, microbes in a mixture or consortium are separated (forexample using physical size and/or cell sorting techniques) followed bydeep DNA or full genome sequencing of the resulting microbes (orsubgroups or subpopulations of microbes). Use of a different microarrayor use of other identification techniques may identify presence ofdifferent microbes (more, fewer, or different microbial taxa or species)than the microarray analysis performed on A1002 described herein, due todifferences in sensitivity and specificity of the analysis techniquechosen. In addition, various techniques (including microarray analysisor PCR DNA analysis) may not detect particular microbes (even if theyare present in a sample), for example if probes capable of detectingparticular microbes are not included in the analysis. In addition, oneof ordinary skill in the art will recognize that microbialclassification and naming may change over time and result inreclassification and/or renaming of microbes.

In other embodiments the disclosed microbial consortia or compositionsinclude, consist essentially of, or consist of 2 or more (such as 5 ormore, 10 or more, 15 or more, 20 or more, or all) of the microbes listedin Table 1.

TABLE 1 Microbes Microbe Exemplary species Desulfococcus spp.Desulfotomaculum spp. Marinobacter spp. Marinobacter bryozoorumNitrosopumilus spp. Azospirillum spp. Bacillus spp. Bacillus subtilis,Bacillus cereus, Bacillus megaterium, Bacillus licheniformis, Bacillusthuringiensis, Bacillus amyloliquefaciens, Bacillus pasteurii, Bacillusoleronius Lactobacillus spp. Lactobacillus acidophilus, Lactobacilluscasei, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillusdelbrueckii, Lactobacillus buchneri Ruminococcus spp. Ruminococcusflavefaciens Aquabacterium spp. Acidisoma spp. Microcoleus spp.Pseudomonas spp. Pseudomonas fluorescens Clostridium spp. Clostridiumbutyricum, Clostridium pasteurianum, Clostridium beijerinckii,Clostridium sphenoides, Clostridium bifermentans Xenococcus spp.Brevibacterium spp. Methanosaeta spp. Lysinibacillus spp. Lysinibacillussphaericus Paenibacillus spp. Paenibacillus chibensis

The consortia or compositions can optionally include one or moreadditional microbes. Additional microbes include, but are not limited toone or more of Deinococcus spp., Leptolyngbya spp., Azotobacter spp.(e.g., Azotobacter vinelandii), Bradyrhizobium spp., Leptospirillum spp.(e.g., Leptospirillum ferrodiazotroph), Paenibacillus spp. (e.g.,Paenibacillus amyloticus), Rhodoferax spp., Halorhabdus spp., Rhizobiumspp. (e.g., Rhizobium japonicum), Bradyrhizobium spp., Micrococcus spp.(e.g., micrococcus luteus), Nitrobacter spp., Nitrosomonas spp.,Nitrococcus spp., Cytophaga spp., Actinomyces spp., Devosia spp.,Streptomyces spp., Streptococcus spp., Lactococcus spp., Proteus spp.(e.g., Proteus vulgaris), Trichoderma spp. (e.g., Trichodermaharzianum), Pediococcus spp. (e.g., Pediococcus pentosaceus),Acetobacter spp. (e.g., Acetobacter aceti), Treponema spp., Candidatusspp., Saccharomyces spp. (e.g., Saccharomyces cerevisiae), Penicillium(e.g., Penicillium roqueforti), Monascus (e.g., Monascus ruber),Aspergillus spp. (e.g., Aspergillus oryzae), Arthrospira (e.g.,Arthrospira platensis), and Ascophyllum spp. (e.g., Ascophyllumnodosum). Suitable additional microbes can be identified by one of skillin the art, for example, based on characteristics desired to be includedin the consortia or compositions.

The disclosed microbial consortia or compositions may include one ormore further components in addition to the microbes, including by notlimited to salts, metal ions, and/or buffers (for example, one or moreof KH₂PO₄, K₂HPO₄, CaCl₂, MgSO₄, FeCl₃, NaMoO₄, and/or Na₂MoO₄), traceelements (such as sulfur, sulfate, sulfite, copper, or selenium),vitamins (such as B vitamins or vitamin K), sugars (such as sucrose,glucose, or fructose), chitin, chitosan, glucosamine, protein, and/oramino acids. Additional components that may also be included in thecompositions include HYTb, HYTc, and/or HYTd, one or more fertilizers(e.g., liquid fertilizer), one or more pesticides, one or morefungicides, one or more herbicides, one or more insecticides, one ormore plant hormones, one or more plant elicitors, or combinations of twoor more of these components.

In some embodiments, the microbial consortia, or a composition includingfive or more microbial species in the microbial consortia describedherein are in a liquid medium (such as a culture or fermentation medium)or inoculum. In other embodiments, the microbial consortia orcomposition including five or more microbial species listed in Table 1are present on a solid or gelatinous medium (such as a culture plate)containing or supporting the microbes.

In yet other embodiments, the microbial consortia or compositionincluding five or more microbial species are present in a dryformulations, such as a dry powder, pellet, or granule. Dry formulationscan be prepared by adding an osmoprotectant (such as a sugar, forexample, trehalose and/or maltodextrin) to a microbial composition insolution at a desired ratio. This solution is combined with dry carrieror absorptive agent, such as wood flour or clay, at the desiredconcentration of microbial composition (such as 2-30%, for example,2.5-10%, 5-15%, 7.5-20%, or 15-30%). Granules can be created byincorporating clay or polymer binders that serve to hold the granulestogether or offer specific physical or degradation properties. Granulescan be formed using rotary granulation, mixer granulation, or extrusion,as a few possible methods. Additional methods for preparing dryformulations including one or more microbial species are known to one ofordinary skill in the art, for example as described in Formulation ofMicrobial Biopesticides: Beneficial Microorganisms, Nematodes and SeedTreatments, Burges, ed., Springer Science, 1998; Bashan, Biotechnol.Adv. 16:729-770, 1998; Ratul et al., Int. Res. J. Pharm. 4:90-95, 2013.

In some examples, compositions including the microbes or microbialconsortia may be maintained at a temperature supporting growth of themicrobe(s), for example at about 25-45° C. (such as about 30-35° C.,about 30-40° C., or about 35-40° C.). In other examples, thecompositions are stored at temperatures at which the microbe(s) are notgrowing or are inactive, such as less than 25° C. (for example, 4° C.,−20° C., −40° C., −70° C., or below). One of skill in the art canformulate the compositions for cold storage, for example by includingstabilizers (such as glycerol). In still further examples, thecompositions are stored at ambient temperatures, such as about 0-35° C.(for examples, about 10-30° C. or about 15-25° C.).

III. Biodegradation Processes

The disclosed microbial consortia or compositions can be used to degradebiological materials, such as chitin-rich materials, for example,aquatic animals or aquatic animal by-products, insects, or fungi. Thus,in some embodiments, disclosed herein are methods including mixing oneor more of the disclosed microbial consortia or compositions with achitin-containing biological material to form a mixture, and fermentingthe mixture. In some embodiments, the methods also include separatingthe mixture into solid, aqueous, and optionally, lipid fractions (FIG.2).

In some embodiments, a biodegradation process disclosed herein includesmixing a microbial consortium (such as A1002, a composition includingsome or all of the microbes in A1002, or a composition including five ormore of the microbial species in Table 1) with one or morechitin-containing biological materials. Chitin-containing biologicalmaterials include, but are not limited to, aquatic animals or aquaticanimal by-products, insects, or fungi. In some examples, thechitin-containing biological material is an aquatic animal, such as anaquatic arthropod (for example, a member of Class Malacostraca). Aquaticarthropods for use in the disclosed methods include shrimp, crab,lobster, crayfish, or krill. In some examples, the entire aquatic animal(such as an aquatic arthropod) or aquatic animal by-products are used inthe biodegradation methods disclosed herein. Aquatic animal by-productsinclude any part of an aquatic animal, such as any part produced byprocessing of the aquatic animal. In some examples, an aquatic animalby-product is all or a portion of an aquatic animal exoskeleton, such asshrimp, crab, crayfish, or lobster shell. In other examples, an aquaticanimal by-product is a part of an aquatic animal, for example, shrimpcephalothoraxes.

In other examples, the chitin-containing biological material includesfungi, such as fungi from Phylum Zygomycota, Basidiomycota, Ascomycota,or Deuteromycota. Particular exemplary fungi include Aspergillus spp.,Penicillium spp., Trichoderma spp., Saccharomyces spp., andSchizosaccharomyces spp. Thus, baker, brewer, and distiller wastestreams can provide sources for chitin-containing biological material.In still further examples, the chitin-containing biological materialincludes insects that contain chitin in their exoskeletons, such asgrasshoppers, crickets, beetles, and other insects. Byproducts of theprocessing of such insects are also contemplated to be sources ofchitin.

The chitin-containing biological material is mixed with a compositionincluding the microbes described in Section II above (such as themicrobial consortium A1002 or other consortium or composition describedin Section II) to form a substantially homogeneous mixture. In someexamples, the chitin-containing biological material is ground, crushed,minced, milled, or otherwise dispersed prior to mixing with the microbesor microbial consortia described herein. In particular examples, themixture contains about 10-50% (such as about 10-20%, about 20-30%, about30-40%, about 25-40%, for example about 25%, about 30%, about 35%, about40%, about 45%, or about 50%) chitin-containing material (such as shrimpheads) (w/v) in inoculum containing about 0.1-5% (such as about 0.1-1%,about 0.5-2%, about 1-2%, about 2-3%, about 0.1%, about 0.2%, about0.3%, about 0.5%, about 0.8%, about 1%, about 1.25%, about 1.5%, about1.75%, about 2%, about 2.5%, about 3%, about 4%, or about 5%) microbes(v/v).

In some examples, the inoculum, chitin-containing biological material,and a sugar (or other carbon source) are mixed together, for example bystirring or agitation. In other examples, one or more of the microbes inthe microbial composition or consortium is optionally activated prior tomixing with the chitin-containing biological material and fermentation.Activation is not required for the methods disclosed herein. Adjustmentsto the time and/or temperature of the fermentation can be made by one ofskill in the art, depending on whether the microbes are activated priorto fermentation. Activation of the microbial composition can be byincubating an inoculum of the microbes with a carbon source (such as asugar, for example, glucose, sucrose, fructose, or other sugar) at atemperature and for a sufficient period of time for the microbes togrow. In some examples, an inoculum of the microbes (such as a microbialconsortium or composition described herein) has a concentration of about0.05-5% v/v (for example, about 0.5-5%, about 0.5-2%, about 1-2%, orabout 2-3%) in a liquid medium. The inoculum is diluted in a solutioncontaining about 0.1-1% sugar (for example, about 0.1-0.5%, about0.1-0.3%, about 0.2-0.6%, or about 0.5-1%, such as about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9%, or about 1%) and incubated at ambient temperatures,for example about 20-40° C. (such as about 20° C., about 25° C., about30° C., about 35° C., or about 40° C.) for about 1-5 days (such as about24 hours, about 48 hours, about 72 hours, about 96 hours, or about 120hours). In other examples, activation of the microbial composition canbe activated by incubating an inoculum of the microbes at a temperatureand for a sufficient period of time for the microbes to grow, forexample, incubation at about 20-40° C. (such as about 25-35° C.) for 12hours to 5 days (such as 1-4 days or 2-3 days). In some non-limitingexamples, the microbes are considered to be activated when the culturereaches an optical density of >0.005 at 600 nm.

After mixing of the chitin-containing biological material and themicrobes or microbial consortium (which are optionally activated), themixture is fermented. In some examples, the pH of the mixture ismeasured prior to fermentation. The pH is adjusted to a selected range(e.g., pH about 3 to about 4 or about 3.5 to 4), if necessary, prior tofermentation. The mixture is incubated at a temperature of about 20-40°C. (for example, about 30°−36° C., such as about 30° C., about 31° C.,about 32° C., about 33° C., about 34° C., about 35° C., about 36° C.,about 37° C., about 38° C., about 39° C., or about 40° C.) for about1-30 days (such as about 3-28 days, about 7-21 days, about 3, 5, 7, 10,14, 16, 20, 24, 28, or 30 days). The mixture is agitated periodically(for example, non-continuous agitation). In some examples, the mixtureis agitated for a period of time every 1-7 days, for example every 1, 2,3, 4, 5, 6, or 7 days. In some non-limiting examples, the fermentationproceeds until the titratable acidity (TTA) is about 3-5% and the pH isabout 4-5.

Following the fermentation, the resulting fermented mixture is separatedinto at least solid and liquid fractions. In some examples, thefermentation is passed from the tank to settling equipment. The liquidis subsequently decanted and centrifuged. In one non-limiting example,the fermented mixture is centrifuged at 1250 rpm (930×g) for 15 minutesat about 5° C. to obtain liquid and lipid (e.g., pigment) fractions. Theliquid (or aqueous) fraction obtained from the biodegradation processcan be stored at ambient temperature. In some non-limiting examples, asugar is added to the liquid fraction, for example at 1-10% v/v.

The liquid fraction may include components such as protein, amino acids,glucosamine, trace elements (such as calcium, magnesium, zinc, copper,iron, and/or manganese), and/or enzymes (such as lactic enzymes,proteases, lipases, and/or chitinases). In some non-limiting examples,the liquid fraction contains (w/v) about 1-5% total amino acids, about3-7% protein, about 0.1-2% nitrogen, less than about 0.2% phosphorus,about 0.5-1% potassium, about 4-8% carbon, about 0.2-1% calcium, lessthan about 0.2% magnesium, less than about 0.2% sodium, and/or about0.1-0.4% sulfur. In additional non-limiting examples, the liquidfraction includes about 0.01-0.2% glucosamine (for example, about 0.1%or less). The liquid fraction also may contain one or more microbes(e.g., from the inoculum used to start the fermentation process) and/ortrace amounts of chitosan or chitin. The liquid fraction is in someexamples referred to herein as “HYTb.”

The solid fraction obtained from the biodegradation process containschitin (for example, about 50-70% or about 50-60% chitin). The solidfraction may also contain one or more of trace elements (such ascalcium, magnesium, zinc, copper, iron, and/or manganese), protein oramino acids, and/or one or more microbes from the inoculum used to startthe fermentation process. The solid fraction is in some examplesreferred to herein as “HYTc.” HYTc is optionally micronized to formmicronized chitin and residual chitin. In some non-limiting examples,the solid fraction contains (w/v) about 9-35% total amino acids, about30-50% crude protein, about 5-10% nitrogen, about 0.3-1% phosphorus,less than about 0.3% potassium, about 35-55% carbon, about 0.5-2%calcium, less than about 0.1% magnesium, about 0.1-0.4% sodium, and/orabout 0.2-0.5% sulfur.

In some examples, a lipid fraction is also separated from the solid andliquid fractions. The lipid fraction is the upper phase of the liquidfraction. The lipid fraction contains compounds such as sterols, vitaminA and/or vitamin E, fatty acids (such as DHA and/or EHA), and in someexamples, carotenoid pigments (for example, astaxanthin). The lipidfraction may be used for a variety of purposes, including but notlimited to production of cosmetics or nutritional products.

In additional embodiments, chitin is fermented with a microbialconsortium (such as A1002 or some or all of the microbes in A1002) or acomposition containing five or more of the microbial species in Table 1.In some examples chitin (such as HYTc, or micronized and/or residualchitin produced as described above) is mixed with a microbial consortiumor composition containing microbes described herein and proteinhydrolyzate (e.g., HYTb), and fermented to form a fermented mixture. Atleast a portion of the chitin in the starting mixture is digested as aresult of the fermentation. In some examples, the mixture is incubatedat a temperature of about 20-40° C. (for example, about 30°-35° C., suchas about 30° C., about 31° C., about 32° C., about 33° C., about 34° C.,about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., orabout 40° C.) for about 1 day to 30 days (such as about 2-28 days, about4-24 days, about 16-30 days, about 10-20 days, or about 12-24 days). Insome examples, the mixture is agitated periodically (for example,non-continuous agitation). In other examples, the mixture iscontinuously agitated. In one non-limiting example, the mixture isagitated for about 1-12 hours daily (such as about 2-8 hours or about4-10 hours). The pH of the fermentation mixture may be monitoredperiodically. In some examples, the pH is optionally maintained at about4-5. In some examples, the fermentation proceeds until Total TitratableAcidity (TTA) is at least about 1-10% (such as about 2-8%, about 4-8%,or about 5-10%).

Following the fermentation, the resulting fermented mixture is separatedinto at least solid and liquid fractions, for example by decanting,filtration, and/or centrifugation. The liquid fraction resulting fromfermentation of HYTb and chitin with the microbial composition is insome examples referred to herein as “HYTd.” In some non-limitingexamples, the liquid fraction contains (w/v) about 0.5-2% total aminoacids, about 3-7% protein, about 0.5-1% nitrogen, less than about 0.1%phosphorus, about 0.4-1% potassium, about 3-7% carbon, less than about0.5% calcium, less than about 0.1% magnesium, less than about 0.3%sodium, and/or about less than about 0.3% sulfur. In addition, HYTdcontains less than about 50% chitin (such as less than about 45%, lessthan about 40%, less than about 35%, or less than about 30% chitin) andless than 2% glucosamine (such as less than about 1.5% or less thanabout 1% glucosamine). In other examples, HYTd contains about 25-50%chitin and about 0.5-2% glucosamine.

IV. Processes for Treating Soil, Plants, and/or Seeds

The disclosed microbial consortia, compositions containing microbes,and/or products disclosed herein (such as HYTb, HYTc, and/or HYTd) canbe used to treat soil, plants, or plant parts (such as roots, stems,foliage, seeds, or seedlings). In some examples, treatment with themicrobial consortia, compositions containing microbes, and/or productsimprove plant growth, improve stress tolerance, and/or increase cropyield.

In some embodiments the methods include contacting soil, plants (such asplant foliage, stems, roots, seedlings, or other plant parts), or seedswith a consortium (such as A1002) or a composition including themicrobes present in one or more of the disclosed microbial consortia orcompositions. The methods may also include growing the treated plants,plant parts, or seeds and/or cultivating plants, plant parts, or seedsin the treated soil.

The microbes are optionally activated before application. In someexamples, activation of the microbes is as described in Section III,above. In other examples, the microbes are activated by mixing 100 partswater and 1 part microbial consortium or composition and incubating atabout 15-40° C. (such as about 20-40° C., about 15-30° C., or about25-35° C.) for about 12 hours-14 days (such as about 1-14 days, 3-10days, 3-5 days, or 5-7 days). The activation mixture optionally can alsoinclude 1 part HYTb, if the microbial consortium or composition is to beapplied in combination with HYTb.

In other embodiments, the methods include contacting soil, plants (orplant parts), or seeds with a product of the disclosed microbialconsortia or compositions, such as HYTb, HYTc, HYTd, or combinationsthereof. In still further embodiments, the methods include contactingsoil, plants, or seeds with a disclosed microbial consortium orcomposition including the disclosed microbes and one or more of HYTb,HYTc, and HYTd (such as one, two, or all of HYTb, HYTc, and HYTd). HYTb,HYTc, and/or HYTd may be separately applied to the soil, plants (orplant parts), and/or seeds, for example sequentially, simultaneously, orsubstantially simultaneously with the disclosed microbial consortia orcompositions containing microbes.

In some examples, the methods further include contacting the soil,plants (or plant part), or seeds with one or more additional componentsincluding but not limited to chitin, chitosan, glucosamine, protein,amino acids, liquid fertilizer, one or more pesticides, one or morefungicides, one or more herbicides, one or more insecticides, one ormore plant hormones, one or more plant elicitors, or combinations of twoor more thereof. The additional components may be included in thecomposition including the microbes or in the microbial consortiadisclosed herein, or may be separately applied to the soil, plants (orplant parts), and/or seeds, for example sequentially, simultaneously, orsubstantially simultaneously with the disclosed microbial consortia orcompositions containing microbes.

In particular embodiments, a microbial consortium or composition iscombined with a liquid fertilizer (for example an aqueous solution orsuspension containing soluble nitrogen). In some examples, the liquidfertilizer includes an organic source of nitrogen such as urea, or anitrogen-containing inorganic salt such as ammonium hydroxide, ammoniumnitrate, ammonium sulfate, ammonium pyrophosphate, ammonium thiosulfateor combinations thereof. Aqua ammonia (20-24.6% anhydrous ammonia) canalso be used as the soluble nitrogen. In some examples, the microbialconsortium or composition is combined with the liquid fertilizer (forexample, mixed with the liquid fertilizer) immediately before use or ashort time before use (such as within 10 minutes to 24 hours before use,for example, about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6hours, 8 hours, 12 hours, 16 hours, 18 hours, or 24 hours before use).

In other examples, the microbial consortium or composition is combinedwith the liquid fertilizer (for example mixed with the liquidfertilizer) at least 24 hours before use (such as 24 hours to 6 months,for example, at least 36 hours, at least 48 hours, at least 72 hours, atleast 96 hours, at least one week, at least two weeks, at least fourweeks, at least eight weeks, or at least 12 weeks before use).

In some examples, the amount of the composition(s) to be applied (forexample, per acre or hectare) is calculated and the composition isdiluted in water (or in some examples, liquid fertilizer) to an amountsufficient to spray or irrigate the area to be treated (if thecomposition is a liquid, such as microbial consortia or compositions,HYTb, or HYTd). In other examples, the composition can be mixed withdiluted herbicides, insecticides, pesticides, or plant growth regulatingchemicals. If the composition to be applied is a solid (such as a dryformulation of microbes, HYTc, chitin, glucosamine, chitosan, or aminoacids), the solid can be applied directly to the soil, plants, or plantparts or can be suspended or dissolved in water (or other liquid) priorto use. In some examples, HYTc is dried and micronized prior to use.

The disclosed microbial compositions (alone or in combination with othercomponents disclosed herein, such as HYTb, HYTc, and/or HYTd) can bedelivered in a variety of ways at different developmental stages of theplant, depending on the cropping situation and agricultural practices.In some examples, a disclosed microbial composition and HYTb are mixedand diluted with liquid fertilizer and applied at the time of seedplanting at a rate of 0.5 to 1 to 2 liters each per acre, oralternatively are applied individually. In other examples, a disclosedmicrobial composition and HYTb are mixed and diluted and applied at seedplanting, and also applied to the soil near the roots at multiple timesduring the plant growth, at a rate of 0.5 to 1 to 2 liters each peracre, or alternatively are applied individually. In still furtherexamples, a disclosed microbial composition and HYTb are diluted anddelivered together through drip irrigation at low concentration asseedlings or transplants are being established, delivered in floodirrigation, or dispensed as a diluted mixture with nutrients in overheador drip irrigation in greenhouses to seedlings or established plants, oralternatively are applied individually. In additional examples, adisclosed microbial composition is added to other soil treatments in thefield, such as addition to insecticide treatments, to enableease-of-use. In other examples, such as greenhouses, a disclosedmicrobial composition and HYTb are used individually or together,combined with liquid fertilizer (such as fish fertilizer) and othernutrients and injected into overhead water spray irrigation systems ordrip irrigation lines over the course of the plant's growth. In onegreenhouse example, a disclosed microbial composition and HYTb are usedtogether, for example, diluted and applied during overhead irrigation orfertigation at a rate of 0.25 to 1 liter at seedling germination,followed by 0.25 to 1 liter mid-growth cycle with fertigation, and final0.25 to 1 liter fertigation 5-10 days end of growth cycle.

In some embodiments, a disclosed microbial composition or consortium andHYTb are applied together or individually (for example sequentially) topromote yield, vigor, typeness, quality, root development, and stresstolerance in crops. In one specific example where the crop is corn, 1 to2 L/acre microbial composition is added in-furrow with liquid fertilizerat seed planting, or applied as a side dress during fertilization afterV3 stage, followed by 0.5 to 2 L/acre of HYTb as a foliar spray after V5stage, added and diluted with herbicides, foliar pesticides,micronutrients, or fertilizers.

In another specific example where the crop is potato, 1 to 3 L/acre ofmicrobial composition is diluted and used either alone or with 1 to 3L/acre of HYTb at tuber planting; this can be followed by subsequentsoil applications of the microbial composition and HYTb beforetuberization, either alone (e.g., sequentially) or together. After plantemergence, potato foliar applications of HYTb at 1 to 2 L/acre can beapplied, either diluted alone or mixed with herbicide, foliar pesticide,micronutrient, or fertilizer treatments, and applied during the growingseason one time, two times, three times, four times, or more.

In yet another specific example where the crop is cotton, 1 to 2 L/acreof microbial composition is applied in-furrow at planting, as a sidedress, or 2×2 (2 inches to side and 2 inches below seed), with orwithout fertilizer. At first white cotton bloom, foliar treatments of0.5 to 2 L/acre HYTb can be applied, diluted alone or combined withother nutrient, herbicide, or pesticidal treatments.

In another particular example where the crop is wheat, the microbialcomposition (1 to 2 L/acre) is applied after winter dormancy (S4 stage)and HYTb applied foliarly (0.5 to 2 L/acre; S4 to S10 stage).

In an example where the crop is sugarcane, one application method uses adisclosed microbial composition and HYTb at 2 to 4 L/acre each, appliedto the soil during cane planting or as a side dress, with foliar HYTbapplied at 1 to 2 L/acre, mixing with water or fertilizers ormicronutrients.

HYTb can be used alone as a foliar treatment in all crops to improvetraits such as plant stress tolerance, vegetative vigor, harvest qualityand yield. In an example where the crop is corn, HYTb can be applied at½ to 1 L/acre, one or multiple times, mixing with water or pesticides orherbicides. In another example, HYTb can be used to treat wheat as afoliar spray, mixed with water or pesticides or herbicides, at a rate of½ to 1 L/acre, applying one or multiple times.

In all crops, HYTc may be added to the soil at a rate of about 0.5-2kg/acre (such as about 0.5 kg/acre, about 1 kg/acre, about 1.5 kg/acre,or about 2 kg/acre) at the time of crop establishment or planting. Inother examples, HYTc is added to a drip irrigation solution of adisclosed microbial composition and HYTb or is added to fertilizationapplications containing a disclosed microbial composition and HYTb ingreenhouses, such as the examples above.

In additional embodiments, HYTd (alone or in combination with themicrobes or other components disclosed herein) is used at about 1-20L/hectare (such as about 1-15 L/hectare, about 3-10 L/hectare, or about3-5 L/hectare). In other examples, HYTd (alone or in combination withthe microbes or other components disclosed herein) is used as a seedtreatment to enhance crop yield and performance (for example, about 1-10L/kg seed, such as about 1-3 L/kg, about 3-5 L/kg, or about 5-10 L/kg).Alternatively, HYTd can be used in the soil (alone or in combinationwith the microbes or other components disclosed herein) at about 1-3L/hectare to increase plant growth, for example to help plants remainproductive under conditions of stress.

In some examples, treatment of soil, seeds, plants, or plant parts witha composition comprising the microbes in a disclosed microbialconsortium increases plant growth (such as overall plant size, amount offoliage, root number, root diameter, root length, production of tillers,fruit production, pollen production, or seed production) by at leastabout 5% (for example, at least about 10%, at least about 30%, at leastabout 50%, at least about 75%, at least about 100%, at least about2-fold, at least about 3-fold, at least about 5-fold, at least about10-fold, or more). In other examples, the disclosed methods result inincreased crop production of about 10-75% (such as about 20-60% or about30-50%) compared to untreated crops. Other measures of crop performanceinclude quality of fruit, yield, starch or solids content, sugar contentor brix, shelf-life of fruit or harvestable product, production ofmarketable yield or target size, quality of fruit or product, grasstillering and resistance to foot traffic in turf, pollination and fruitset, bloom, flower number, flower lifespan, bloom quality, rooting androot mass, crop resistance to lodging, abiotic stress tolerance to heat,drought, cold and recovery after stress, adaptability to poor soils,level of photosynthesis and greening, and plant health. To determineefficacy of products, controls include the same agronomic practiceswithout addition of microbes, performed in parallel.

The disclosed methods can be used in connection with any crop (forexample, for direct crop treatment or for soil treatment prior to orafter planting). Exemplary crops include, but are not limited toalfalfa, almond, banana, barley, broccoli, canola, carrots, citrus andorchard tree crops, corn, cotton, cucumber, flowers and ornamentals,garlic, grapes, hops, horticultural plants, leek, melon, oil palm,onion, peanuts and legumes, pineapple, poplar, pine and wood-bearingtrees, potato, raspberry, rice, sesame, sorghum, soybean, squash,strawberry, sugarcane, sunflower, tomato, turf and forage grasses,watermelon, wheat, and eucalyptus.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

Example 1 Microbial Consortium A1002

This example describes production of microbial consortium A1002.

A1002 was produced from a seed batch of microbes that originally werederived from fertile soils and additional microbes (such as Bacillusspp.) (see, e.g., U.S. Pat. No. 8,748,124, incorporated herein byreference). The “seed” culture was mixed with a suspension containing5.5% w/w whey protein and 1.2% w/w yogurt in water (“C vat”) and asuspension containing 0.1% w/w spirulina and 0.1% w/w kelp extract inwater (“A vat”). The A vat and C vat suspensions were each individuallyprepared 3 days before mixing with the seed culture and incubated atambient temperature. The seed culture, C vat, and A vat were mixed at aproportion of about 81:9:9. After mixing, a suspension of additionalcomponents containing about 70% v/v molasses, 0.5% v/v HYTb, 0.003% w/vArabic gum, and 0.02% w/v brewer's yeast (S. cerevisiae) were mixed withthe mixture of the seed culture, C vat, and A vat, and additional waterat a ratio of about 16:34:50. The mixture was fermented for about 7 daysat ambient temperature (about 19-35° C.). After 7 days, the tanks wereaerated for 30 minutes every other day. Additional water was added(about 10% more v/v) and fermentation was continued under the sameconditions for about 10 more days. Additional water was added (about 4%more v/v) and fermentation was continued for about 7 more days, at whichtime samples were collected for analysis and deposit with the ATCC.A1002 was subsequently stored in totes at ambient temperature.

Example 2 Analysis of Microbes in A1002 by Plating

This example describes analysis of microbes present in A1002 byreplicate plating under aerobic and anaerobic conditions.

Samples (50 mL) were collected from an aerated tote of A1002 (stirredwith a stainless steel mixing paddle at 120 rpm for 8 minutes) using asanitized handheld siphon drum pump. On day 1, the sample was vortexed(e.g., 60 seconds at 2000 rpm) to ensure even distribution of microbes.In a tube with 9.8 mL sterile water, 0.1 mL of A1002 sample and 0.1 mLof HYTb were added (10⁻² dilution). The tube was incubated at 35° C. for72 hours without shaking. After 72 hours (day 3), the tube was brieflyvortexed and a series of 10-fold dilutions in sterile water was prepared10⁻³ to 10⁻⁹ dilutions).

Each dilution was plated (100 μL) on a Nutrient Agar plate (for aerobicmicroorganism culture) and a Standard Methods Agar plate (for anaerobicmicroorganism culture), with 3 replicates for each. Nutrient Agar plateswere cultured at 27° C. for 48 hours. Standard Methods Agar plates wereincubated at 35° C. for 72 hours in an anaerobic chamber. After theincubation, for each culture, a dilution that yielded less than 100colonies was selected. For the selected dilution all of the colonies oneach of the replicate plates were counted and CFU/mL calculated. A1002yielded 4.6×10⁷ CFU/mL under aerobic conditions and 4.0×10⁷ CFU/mL underanaerobic conditions.

Example 3 Analysis of Microbes in A1002 by Microarray

This example describes microarray analysis of microbes present in A1002.

A sample of A1002 was analyzed by Second Genome (South San Francisco,Calif.) using the G3 PhyloChip™ Assay. DNA was isolated from the sampleusing PowerSoil® DNA isolation kit (Mo Bio Laboratories, Inc., Carlsbad,Calif.) according to the manufacturer's instructions. 16S rRNA wasamplified (35 PCR cycles) using Genes were amplified using thedegenerate forward primer 27F.1 (AGRGTTTGATCMTGGCTCAG; SEQ ID NO: 1) andthe non-degenerate reverse primer 1492R (GGTTACCTTGTTACGACTT; SEQ ID NO:2). The amplification products were concentrated using a solid-phasereversible immobilization method and quantified by electrophoresis usingan Agilent 2100 Bioanalyzer®. PhyloChip Control Mix™ was added to eachamplified product. The amplicons were fragmented, biotin labeled, andhybridized to the PhyloChip™ G3 array, which includes >1.1 millionprobes targeting about 55,000 individual microbial taxa, with multipleproves per operational taxonomic unit (OTU). The arrays were washed,stained, and scanned using a GeneArray® scanner (GeneChip® MicroarrayAnalysis Suite, Affymetrix).

Approximately 330 billion molecules were assayed and analyzed usingSecond Genome's PhyloChip processing software. A series of perfect match(PM) and mis-match (MM) probes sets gave off a florescence intensity(FI) which were captured as pixels in an image and collected as aninteger value. The software then made adjustments for backgroundflorescence and noise estimation and rank-normalized the results. Theresults were then used as input to empirical probe-set discovery. Theempirical OUT tracked by a probe set was then taxonomically annotatedagainst the May 2013 release of Greengenes 16S rRNA gene database(greengenes.lbl.gov) from the combination of 8-mers contained in allprobes of the set. The taxa were then identified by the standardtaxonomic name or with a hierarchical taxon identifier.

After the taxa were identified for inclusion in analysis, the valuesused for each taxa-sample were populated in two distinct ways. In thefirst case, a relative abundance metric was used to rank the abundanceof each taxa relative to the others. The second case used a binarymetric or presence/absence score to determine whether each taxon wasactually in the sample.

The data from the microarray analysis were also used to select microbesfor inclusion in the compositions described herein (such as the microbeslisted in Table 1 and elsewhere herein). The microbes (taxa, genus, orspecies) were ranked in order of relative abundance and microbes wereselected based on desired characteristics.

Example 4 Analysis of Microbes in A1002 by Sequencing

This example describes exemplary methods for analysis of microbes inA1002 by sequencing 16S rDNA. One skilled in the art will appreciatethat methods that deviate from these specific methods can also be usedfor successful sequencing and analysis of microbes in A1002.

Genomic DNA was extracted from a sample of A1002. 16S rDNA was amplifiedby PCR and sequenced, for example using MICROSEQ ID microbialidentification system (Applied Biosystems/Life Technologies, GrandIsland, N.Y.). Sequencing data was analyzed using SHERLOCK DNA software(MIDI Labs, Newark, Del.). Purified isolates were identified and arelisted in Table 2. A species level match was assigned if the % genericdifference (% GD) between the unknown and the closest match was lessthan the approximate average % GD between species within that particulargenetic family, which is usually 1%. A genus level match was assignedwhen the sequence did not meet the requirements for a species levelmatch, but still clustered within the branching of a well-defined genus(GD greater that 1% and less than about 3%).

TABLE 2 Microbes identified in A1002 by 16S rDNA sequencing ConfidenceSample Microbe Level % GD Base pairs 1a Bacillus oleronius Species 0.37537 1b Bacillus thuringiensis Species 0.28 537 1c Lysinibacillussphaericus Genus 1.96 536 2a Lactobacillus buchneri Species 0.18 561 2cBacillus oleronius Species 0.19 537 2d Paenibacillus chibensis Genus 3.9538 3a Lactobacillus buchneri Species 0.27 561 4b Bacillus oleroniusSpecies 0.37 537 5a Lactobacillus buchneri Species 0.36 561

Example 5 Biodegradation of Chitin-Containing Materials

This example describes exemplary methods for biodegradation ofchitin-containing biological materials using the microbial consortiumA1002. However, one skilled in the art will appreciate that methods thatdeviate from these specific methods can also be used for successfulbiodegradation of chitin-containing biological materials.

Shrimp by-products are obtained from shrimp processing plants andtransported in closed, chilled containers. After inspection of the rawmaterial quality, the shrimp by-products are homogenized to reduceparticle size to about 3-5 mm. Pre-activated A1002 microbial cultures(about 0.2-100 mL/L) and sugar (about 5 g/L) are mixed with thehomogenized shrimp by-product (about 50 g/L) and agitated until themixture is homogeneous. With continuous agitation, the temperature ismaintained at ambient temperature (about 19-35° C.) and the pH isadjusted to 3.5-4.0 with citric acid. The mixed ingredients aretransferred into a sanitized fermentation tank (25,000 L) and fermentedat 30-36° C. for 120 hrs. Agitation is applied for 30 minutes at leasttwo times a day. During the fermentation process, the pH is monitored,and the total titratable acidity (TTA, %) is determined by titrationwith 0.1 N NaOH. The fermentation is stopped when the TTA is about 3.5%and/or the pH is about 4-5.

The fermented cultures are fed to a continuous decanter. The separatedsolid layer from the decanting step is subject to centrifugation toremove the lipid layer. The purified liquid (HYTb) is mixed with sugar(such as molasses, 10% v/v), then stored in holding tanks or dispensedto totes. The solid materials from the decanting step are dried withsuperheated air at 120° C. until their moisture content is below 8%,then ground to 200 mesh. The dried product (HYTc) is packaged in bags orsacks.

Example 6 Biodegradation of Chitin

This example describes exemplary methods for biodegradation of chitinusing the microbial consortium A1002. However, one skilled in the artwill appreciate that methods that deviate from these specific methodscan also be used for successful biodegradation of chitin.

A1002 microbial culture is pre-activated with sugar (about 2.5 g/L) in a10,000 L tank for three days. The activated inoculum is mixed withprotein hydrolysate such as HYTb (about 500 mL/L) and chitin (HYTc e.g.,produced as described in Example 5). The mixture is gently mixed for 1hour to achieve complete homogenization. The mixture is fermented for 20days at ambient temperature (e.g., about 19-35° C.) with agitation forabout 8 hours daily and pH monitoring (pH 4.0-5.0). Samples may becollected periodically, for instance every two days, for quantificationof glucosamine and optionally chitosan. After fermentation is complete,the mixture is filtered through a filter that retains particles of 300mesh, primarily the remaining chitin. The filtrate is retained andbottled after product characterization.

Example 7 Treatment of Field Corn with Microbial Compositions

This example describes a representative method for obtaining increasedcorn crop yield, using a microbial consortium. One skilled in the artwill appreciate that methods that deviate from these specific methodscan also be used for increasing crop yield.

Treatment of field corn with a microbial composition prepared similarlyto A1002, or with HYTb, showed a strong increase in final harvestableyield. All agronomic practices of fertilization, cultivation, weedcontrol, and pest control, were identical and side-by-side for themicrobial composition- or HYTb-treated plots (Test) and control (Check)plots.

Trial 1 evaluated yield after the microbial composition was added to thetypical nitrogen side dress (1 L/acre microbial composition; 32 UANliquid fertilizer; Test) compared to non-treated control (Check),applied at V2 stage. In two large-scale, replicated strip trials (1 acretotal), yield in the Test strips were 8% to 10% higher than parallelcontrol strips (Check) (FIG. 4A).

Trial 2 demonstrated that both in-furrow application and addition in theside dress were equally effective for increasing corn yields. In a 1acre strip trial, large plots were treated with the microbialcomposition added in-furrow, during seed planting (1 L/acre) or at V2stage as a side dress (3 gal NPK liquid fertilizer, 1 litermicronutrient mix). Both application methods showed Test strips hadabout a 5% increase in yield, about 10 Bu/acre compared to controls(FIG. 4B). Adding a commercial blend of 10% humic acid/biostimulant tothe Test (Actuate) in-furrow offered the same 5% yield as microbialcomposition addition alone compared to non-treated control (FIG. 4B).

Trial 3 demonstrated that addition of nitrogen-stabilization productseither unaffected or slightly boosted the yield enhancing effect of themicrobial composition in corn and further validated the consistent boostin yield of the microbial composition delivered either in-furrow ormixed in the side dress (FIG. 4C). In a 1 acre strip trial, bothin-furrow and side dress treatments offered a 3% yield boost (8 Bu/acre)over control (Check). Addition of Actuate caused a slight yield increase(4% boost in yield, 9 Bu/acre higher than control). Addition ofnitrogen-stabilization products, Instinct or N-Kress, caused either noeffect (modest 2.5% yield boost for Instinct) or a slightly higher boostin yield (4.6% yield increase for N-Kress, 11 Bu/acre higher thancontrol).

Trial 4 demonstrated that HYTb delivered in-furrow also boosted yieldover control plots. In a 20 acre trial, HYTb was added to the in-furrowfertilizer/nutrient mix (1 L/acre). Compared to parallel control acreage(Check), HYTb-treated acres offered a 3.5% (7 Bu/acre) yield increase(FIG. 4D).

Trial 5 demonstrated that, when evaluated in a replicated plot designtrial, a single soil inoculation of corn with the microbial compositionat 1 L/acre in furrow at V6 stage, delivered with 28% nitrogenfertilizer via drip irrigation, provided a 14% increase yield over theuntreated control across five replicated plots (FIG. 4E).

Trial 6 showed that HYTb, when used alone as a foliar treatment in corn,also provided a 9.5% yield increase when compared to the untreatedcontrol when tested in a randomized, replicated plot design trial. HYTbwas foliar sprayed over two applications of 1 L/acre each application,at the V8 stage and VT stages (FIG. 4F).

Trial 7 was also a randomized and replicated plot design trial in corn,performed under water stress conditions. In this study, the amount ofirrigation was limited to 11 inches of water versus the appropriatelywatered plots that received 17 inches of irrigation. A single 1 L/acretreatment of microbial composition, delivered at stage V6 with 28%nitrogen fertilizer via drip irrigation (Treated), produced a 38% yieldincrease over plots treated with fertilizer alone (untreated Check). Theharvest increase observed with microbial composition treatmentrepresents a potential of 31 Bu/acre higher yield (FIG. 4G).

Example 8 Treatment of Wheat with Microbial Compositions

This example describes a representative method for obtaining increasedwheat crop yield, using a microbial consortium. One skilled in the artwill appreciate that methods that deviate from these specific methodscan also be used for increasing crop yield.

Treatment of wheat with a microbial composition prepared similarly toA1002, or with HYTb, showed a strong increase in final harvestableyield. All agronomic practices of fertilization, cultivation, weedcontrol, and pest control, were identical and side-by-side for themicrobial composition- or HYTb-treated plots (Test) and control (Check)plots.

Trial 1 showed a strong increase in wheat yield promoted by soilapplication of the microbial composition. In this 80 acre trial, themicrobial composition was added at a rate of 1 L/acre to the top dressfertilizer mix at stage S4. Harvest yields demonstrated an 11% (10Bu/acre) yield increase with use of the microbial composition (FIG. 5A).

Trial 2 compared three large trials in the same geographic area,totaling 271 acres of microbial composition-treated (test) and 354 acresof parallel untreated wheat (control). All trials were performed thesame, with microbial composition (1 L/acre) added to the top dressfertilizer mix and applied at wheat growth stage S4. Relative toparallel control acres on the same farm, the treated wheat gave higheryields, ranging from an increase of 6% to 17% to 36% higher yields, witha three farm average of about 16% increase in yield (FIG. 5B).

Trial 3 evaluated microbial composition and HYTb treatment of wheat incombination and found that the combination enhanced yield. In a largepivot trial (129 acres), microbial composition was applied pre-plant ata rate of 1 L/acre, incorporated with normal nutritional program, andfollowed by pivot delivery of HYTb as a foliar spray (1 L/acre) plusherbicide at wheat growth stage S6. Compared to untreated control(Check), the treated acreage gave a 10% higher yield (14 Bu/acre) thancontrol acreage (FIG. 5C). Further, typical wheat plants from thetreated plots had visibly more roots than untreated controls (FIG. 5D).

Example 9 Treatment of Tomato with Microbial Compositions

This example describes a representative method for obtaining increasedtomato crop yield, using a microbial consortium. One skilled in the artwill appreciate that methods that deviate from these specific methodscan also be used for increasing crop yield.

Treatment of tomato with a microbial composition prepared similarly toA1002 showed a strong increase in final harvestable yield. All agronomicpractices of fertilization, cultivation, weed control, and pest control,were identical and side-by-side for both the microbialcomposition-treated (Test) and control (Check) plots.

Trial 1 evaluated microbial composition treatment of tomato applied at 1L/acre with one application at transplant (in transplant water) followedby application by drip irrigation every three weeks (four times). In a10 acre test plot compared to a 10 acre control plot, the treatedacreage gave about 8% higher yield than control (FIG. 6A).

Trial 2 evaluated microbial composition treatment of tomato applied at 1L/acre by drip irrigation every three weeks (five times). In a 49.6 acretest plot compared to a 4.45 acre control plot, the treated acreage gaveabout 9% higher yield than control (FIG. 6B).

Trial 3 evaluated microbial composition treatment of tomato applied at 1L/acre with one application at transplant (in transplant water) followedby application by drip irrigation every three weeks (three times). In a15.6 acre test plot compared to a 73.2 acre control plot, the treatedacreage gave about 29% higher yield than control (FIG. 6C).

Trial 4 evaluated microbial composition treatment of tomato applied at 1L/acre with by drip irrigation every three weeks (four times). In an 8.7acre test plot compared to a 6.57 acre control plot, the treated acreagegave decreased yield compared to control (FIG. 6D). However, the trialwas affected by severe disease pressure (Fusarium) which likely affectedthe outcome of the trial. In addition, this trial was a relatively smallplot size and also included different crop varieties in the treatment.

Trial 5 evaluated microbial composition treatment of tomato applied at 1L/acre in combination with fertilizer treatment. One application was attransplant with 8-7-7, followed by application by drip irrigation everythree weeks (three times) with UAN. In a 33.3 acre test plot compared toa 16.45 acre control plot, the treated acreage gave about 5% higheryield than control (FIG. 6E).

Example 10 Treatment of Sunflower with Microbial Compositions

This example describes a representative method for obtaining increasedsunflower crop yield, using a microbial consortium. One skilled in theart will appreciate that methods that deviate from these specificmethods can also be used for increasing crop yield.

Treatment of sunflower crop with a microbial composition preparedsimilarly to A1002 showed a strong increase in final harvestable yield.All agronomic practices of fertilization, cultivation, weed control, andpest control, were identical and side-by-side for both the microbialcomposition-treated (Test) and control (Check) plots.

This trial evaluated microbial composition treatment of sunflowerapplied at 1 L/acre by drip irrigation 30 days and 60 dayspost-planting. In a 93.5 acre test plot compared to a 97.13 acre controlplot, the treated acreage gave about 50% higher yield than control (FIG.7). In addition, the treatment resulted in increased germination rates.

Example 11 Treatment of Rice with Microbial Compositions

This example describes a representative method for obtaining increasedrice crop yield, using a microbial consortium. One skilled in the artwill appreciate that methods that deviate from these specific methodscan also be used for increasing crop yield.

Treatment of rice with a microbial composition prepared similarly toA1002 showed a strong increase in final harvestable yield. All agronomicpractices of fertilization, cultivation, weed control, and pest control,were identical and side-by-side for both the microbialcomposition-treated (Test) and control (Check) plots.

This trial evaluated microbial composition treatment of rice applied at1 L/acre with aqua ammonia. In a 61.8 acre test plot compared to a 100.7acre control plot, the treated acreage gave about 6% higher yield thancontrol (FIG. 8).

Example 12 Treatment of Soybean with Microbial Compositions

This example describes a representative method for obtaining increasedsoybean crop yield, using a microbial consortium. One skilled in the artwill appreciate that methods that deviate from these specific methodscan also be used for increasing crop yield.

Treatment of soybean with a microbial composition prepared similarly toA1002, or with HYTb, showed a strong increase in final harvestableyield. All agronomic practices of fertilization, cultivation, weedcontrol, and pest control, were identical and side-by-side for themicrobial composition- or HYTb-treated plots (Test) and control (Check)plots.

Trial 1 showed an increase in soybean yield promoted by application ofHYTb at 1 L/acre, applied with fungicide. In two one acre tests, thetreated acreage gave about 5% increased yield compared to control (FIG.9A).

Trial 2 evaluated microbial composition treatment or microbialcomposition plus HYTb treatment of soybean applied at 1 L/acre by foliarand side dress application. The treated acreage had reduced yieldcompared to control (FIG. 9B). However, the trial was affected by smallplot size combined with wildlife problems (deer nested and consumed thebeans before harvest).

Trial 3 showed an increase in soybean yield promoted by application ofHYTb at 0.5 L/acre, applied with fungicide by foliar application. In a60 acre test plot compared to a 26.48 acre control plot, the treatedacreage gave about 12% increased yield compared to control.

Example 13 Treatment of Strawberry with Microbial Compositions

This example describes a representative method for obtaining increasedstrawberry crop yield, using a microbial consortium. One skilled in theart will appreciate that methods that deviate from these specificmethods can also be used for increasing crop yield.

Treatment of strawberry with a microbial composition prepared similarlyto A1002 plus HYTb showed increases in final harvestable yield. Allagronomic practices of fertilization, cultivation, weed control, andpest control, were identical and side-by-side for both the treated(Test) and control (Check) plots.

An increase in cumulative marketable production was promoted byapplication of microbial composition and HYTb applied by dripirrigation. In these five independent trials, the Sabrina variety wasevaluated in the Huelva region of Spain. One week prior to plantlettransplantation in the raised bed plots, 2 L of the microbialcomposition plus 4 L HYTb were diluted in water and added to the dripirrigation per hectare, with the same application rate performed atweeks 2, 4, and 6 post-planting. At weeks 3, 5, and 7, diluted microbialcomposition was added at a rate of 1 L/ha and diluted HYTb at a rate of2 L/ha. From week 9 to the end of the harvest season, diluted microbialcomposition and HYTb were added at rates of 1 L/ha each. In all fivetrials, the treatment boosted yield from 5% to 11% above parallelnon-treated plots, for an average of about an 8% yield increase acrossall five trials (FIG. 10).

Example 14 Treatment of Beetroot with Microbial Compositions

This example describes a representative method for obtaining increasedbeetroot crop yield, using a microbial consortium. One skilled in theart will appreciate that methods that deviate from these specificmethods can also be used for increasing crop yield.

Treatment of beetroot with a microbial composition prepared similarly toA1002 plus HYTb showed increases in final harvestable yield. Allagronomic practices of fertilization, cultivation, weed control, andpest control, were identical and side-by-side for both the treated(Test) and control (Check) plots.

An increase in average harvested head weight was promoted by applicationof microbial composition (2 L/acre) and HYTb (2 L/acre) applied by dripirrigation and HYTb (1 L/acre) by foliar application. In an 8 acre testplot compared to a 9 acre control plot, the treated acreage gave about2.2-fold higher yield than control (FIG. 11).

Example 15 Treatment of Green Cabbage with Microbial Compositions

This example describes a representative method for obtaining increasedgreen cabbage crop yield, using a microbial consortium. One skilled inthe art will appreciate that methods that deviate from these specificmethods can also be used for increasing crop yield.

Treatment of green cabbage with a microbial composition preparedsimilarly to A1002, or with HYTb, showed a strong increase in finalharvestable yield. All agronomic practices of fertilization,cultivation, weed control, and pest control, were identical andside-by-side for the microbial composition- or HYTb-treated plots (Test)and control (Check) plots.

The trials showed an increase in cabbage yield promoted by applicationof microbial composition (2 L/acre) and HYTb (2 L/acre) applied by dripirrigation and HYTb (1 L/acre) by foliar application. Cabbages wereharvested in two cycles, as represented by the “first cut” harvest ofcabbage heads and the later “second cut” of cabbage heads. As shown inFIG. 12A, in a 10.9 acre test plot compared to a 14.9 acre control plot,the treated acreage gave about 18% higher yield than control (first cut)and about 31% higher yield than control (second cut). As shown in FIG.12B, in a 3.7 acre test plot compared to a 1.5 acre control plot, thetreated acreage gave about 61% higher yield than control (first cut) andabout 64% higher yield than control (second cut).

Example 16 Seed and Tuber Treatment with HYTd

This example describes a representative method for obtaining increasedwheat and potato crop yield using pre-treatment of the seed or seedtubers with HYTd. One skilled in the art will appreciate that methodsthat deviate from these specific methods can also be used for increasingcrop yield.

Treatment of wheat seed or potato seed tubers prior to planting withHYTd prepared using a microbial consortium similar to A1002 showedincreases in final harvestable yield. All agronomic practices offertilization, cultivation, weed control, and pest control, wereidentical and side-by-side for both the treated (Test) and control(Check) plots.

For wheat, seed was treated in a diluted suspension of HYTd, diluted ata rate of 3 mL of HYTd in water per kg of seed. After coating seed andallowing air drying, treated seed was planted and compared to identicalplots of untreated seed. One acre parallel field plots showed about 22%increase in wheat harvested yield (Table 3).

Potato seed treatment was performed by diluting HYTd in water andtreating potato seed at a rate of 1 mL per kg of seed. After air drying,the treated potato seed was planted in parallel with untreated controlseed in 1200 meter, replicated plots. HYTd treated potato seed increasedpotato yield 32% to 35% in two separate trials (Table 4).

TABLE 3 Yield from HYTd treated wheat seed Weight of Dose Straw weight/5m² grains 5 m² Yield Treatment (ml/kg seed) area (kg) area (kg)(kg/acre) HYTd 3.00 9.8 2.6 1980 Untreated N/A 5.5 1.7 1610

TABLE 4 Yield from HYTd treated potato seed Dose Weight of Final (ml/kgNumber of tubers/m² Yield % increase Treatment seed) tubers/plant (kg)(kg/acre) tuber yield Trial 1 HYTd 1.00 11 3.15 12448 32 Untreated N/A 61.68 9440 0 Trial 2 HYTd 1.00 8 2.52 10720 35 Untreated N/A 5 1.47 79320

Example 17 Increased Stress Tolerance in Potato

This example describes a representative method for obtaining increasedpotato tuber quality by treating with a microbial composition similar toA1002 and HYTb during growth under stressful field conditions.

Russet Burbank variety potato was grown under conventional conditions ina replicated plot trial (four replicates) and either treated (microbialcomposition plus HYTb at 1 L each per acre at planting, in furrow,followed by two foliar spray applications of HYTb at 1 L/acre at 55 daysand again 85 days after planting) or untreated (control). Russet Burbankvariety is prone to lower quality under water, heat, or nutrient stress.In this trial, the microbial composition and HYTb treatment enhancedtolerance to a stress-induced quality defect called hollow heart. Plotstreated with microbial composition had an incidence of 1.68% ofharvested tubers with hollow heart compared to the control with 8.35%hollow heart defects (Table 5).

TABLE 5 Potato hollow heart quality defects Yield Hollow Heart Treatment(kg/acre) percentage Untreated (control) 32,181 8.35% Microbialcomposition 32,636 1.68%* plus HYTb *p < 0.01 compared to untreated

Example 18 Cucumber Vigor Assay

Rapid plant-based functional assays can be used to quickly evaluateplant response to new microbial compositions. Using a cucumber vigor andplant growth assay, this example demonstrates that A1002 enhances therate of plant leaf growth and expansion.

After pre-germination of cucumber seedlings in nutrient-soaked rolledgermination paper for four days, staged and synchronized plants weretreated with a diluted mixture of liquid fertilizer and microbialconsortium. Plantlets were transplanted into prepared soilless growthmedium pre-treated with fertilizer and the tester solution. Themicrobial composition A1002 was diluted 1:2000 in a nutrient fertilizermedia. As control treatment, an equivalent amount of water added tonutrient media was compared. At least 16 plants of each treatment grownin pots, including control plants, were randomized in flats, and grownunder defined growth conditions, controlling for temperature and light.After 18 days, the Leaf Area Index (LAI) of the first true leaf of eachplant was measured. The total plant wet weight was also recorded. Thedata was analyzed by One-way ANOVA (Analysis Of Variance) and withpost-hoc Tukey test to compare samples within the experiment.

At day 18, the first leaf LAI rating promoted by A1002 treatment wassignificantly greater than the control (FIG. 13).

In addition to, or as an alternative to the above, the followingembodiments are described:

Embodiment 1 is directed to a composition comprising the microbes inATCC deposit PTA-121751 (A1002).

Embodiment 2 is directed to a composition comprising five or moremicrobial species selected from Bacillus spp., Azospirillum spp.,Pseudomonas spp., Lactobacillus spp., Desulfococcus spp.,Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp.Ruminococcus spp., Aquabacterium spp., Acidisoma spp., Microcoleus spp.,Clostridium spp., Xenococcus spp., Brevibacterium spp., and Methanosaetaspp.

Embodiment 3 is directed to a composition comprising ten or moremicrobial species selected from Bacillus spp., Azospirillum spp.,Pseudomonas spp., Lactobacillus spp., Desulfococcus spp.,Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp.Ruminococcus spp., Aquabacterium spp., Acidisoma spp., Microcoleus spp.,Clostridium spp., Xenococcus spp., Brevibacterium spp., and Methanosaetaspp.

Embodiment 4 is directed to a composition comprising each of Bacillusspp., Azospirillum spp., Pseudomonas spp., Lactobacillus spp.,Desulfococcus spp., Desulfotomaculum spp., Marinobacter spp.,Nitrosopumilus spp. Ruminococcus spp., Aquabacterium spp., Acidisomaspp., Microcoleus spp., Clostridium spp., Xenococcus spp.,Brevibacterium spp., and Methanosaeta spp.

Embodiment 5 is directed to a composition of any one of embodiments 1 to4, further comprising one or more of chitin, chitosan, glucosamine, andamino acids.

Embodiment 6 is directed to a method comprising:

-   -   mixing a chitin-containing biological source with the        composition of any one of embodiments 1 to 5 to form a mixture;    -   fermenting the mixture; and    -   separating the fermented mixture into solid, aqueous, and lipid        fractions.

Embodiment 7 is directed to the method of embodiment 6, wherein thechitin-containing biological source comprises an aquatic animal oraquatic animal by-product, an insect, or a fungus.

Embodiment 8 is directed to the method of embodiment 7, wherein theaquatic animal is an aquatic arthropod.

Embodiment 9 is directed to the method of embodiment 8, wherein theaquatic arthropod is shrimp, crab, or krill.

Embodiment 10 is directed to the aqueous fraction made by the method ofany one of embodiment 6 to 9.

Embodiment 11 is directed to a solid fraction made by the method of anyone of embodiments 6 to 9.

Embodiment 12 is directed to a method comprising contacting soil,plants, or plant parts with the composition of any one of embodiments 1to 5.

Embodiment 13 is directed to the method of embodiment 12, furthercomprising contacting the soil, plants, or plant parts with one or moreof chitin, chitosan, glucosamine, and amino acids.

Embodiment 14 is directed to the method of embodiment 12 or 13, furthercomprising contacting the soil, plants, or plant parts with the aqueousfraction of claim 10 or the solid fraction of claim 11.

Embodiment 15 is directed to the method of any one of embodiments 12 to14, further comprising contacting the soil, plants, or plant parts witha liquid fertilizer.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

1. A composition comprising five or more microbial species selected fromBacillus spp., Azospirillum spp., Pseudomonas spp., Lactobacillus spp.,Desulfococcus spp., Desulfotomaculum spp., Marinobacter spp.,Nitrosopumilus spp. Ruminococcus spp., Aquabacterium spp., Acidisomaspp., Microcoleus spp., Clostridium spp., Xenococcus spp.,Brevibacterium spp., and Methanosaeta spp.
 2. The composition of claim1, comprising ten or more microbial species selected from Bacillus spp.,Azospirillum spp., Pseudomonas spp., Lactobacillus spp., Desulfococcusspp., Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp.Ruminococcus spp., Aquabacterium spp., Acidisoma spp., Microcoleus spp.,Clostridium spp., Xenococcus spp., Brevibacterium spp., and Methanosaetaspp.
 3. The composition of claim 2, comprising each of Bacillus spp.,Azospirillum spp., Pseudomonas spp., Lactobacillus spp., Desulfococcusspp., Desulfotomaculum spp., Marinobacter spp., Nitrosopumilus spp.Ruminococcus spp., Aquabacterium spp., Acidisoma spp., Microcoleus spp.,Clostridium spp., Xenococcus spp., Brevibacterium spp., and Methanosaetaspp.
 4. The composition of claim 1, further comprising one or more ofchitin, chitosan, glucosamine, and amino acids.
 5. A method comprising:mixing a chitin-containing biological source with the composition ofclaim 1 to form a mixture; fermenting the mixture; and separating thefermented mixture into solid, aqueous, and lipid fractions.
 6. Themethod of claim 5, wherein the chitin-containing biological sourcecomprises an aquatic animal or aquatic animal by-product, an insect, ora fungus.
 7. The method of claim 6, wherein the aquatic animal is anaquatic arthropod.
 8. The method of claim 7, wherein the aquaticarthropod is shrimp, crab, or krill.
 9. The aqueous fraction made by themethod of claim
 5. 10. The solid fraction made by the method of claim 5.11. A method comprising contacting soil, plants, or plant parts with thecomposition of claim
 1. 12. The method of claim 13, further comprisingcontacting the soil, plants, or plant parts with one or more of chitin,chitosan, glucosamine, and amino acids.
 13. (canceled)
 14. The method ofclaim 11, further comprising contacting the soil, plants, or plant partswith a liquid fertilizer.
 15. The method of claim 11, further comprisingcontacting the soil, plants, or plant parts with one or more pesticides,one or more fungicides, one or more herbicides, one or moreinsecticides, one or more plant hormones, one or more plant elicitors,or combinations of two or more thereof.
 16. A method comprising:contacting soil, plants, or plant parts with the composition of claim 1and with an aqueous fraction or a solid fraction, wherein the aqueousfraction and the solid fraction are made by: mixing a chitin-containingbiological material with the composition of to form a mixture;fermenting the mixture; and separating the fermented mixture into thesolid fraction, the aqueous fraction, and a lipid fraction.